There are at present various screening methods for genotoxic and/or toxic compounds using microorganisms. The Ames test (Maron and Ames, 1983) appears to be the most widely used test for toxic compounds and is as such recommended worldwide. It however takes about three days for completion of the test with the obvious concomitant disadvantages.
Some short term methods have been introduced wherein the SOS response caused by DNA damage is measured as an amount of xcex2-galactosidase expressed by the lacZ gene positioned downstream of the umuD,C or sfiA stress induced promoters that are SOS regulated. These tests are known respectively as the umu test (Oda et al. 1985) using Salmonella typhimurium as host microorganism and SOS chromotest (Quillardat et al., 1982) using E. coli as host microorganism. In the umu test and SOS chromotest the host microorganism is cultured in the presence of the sample to be tested and subsequently the host microorganism is disrupted. A substrate for xcex2-galactosidase is then added and the ensuing reaction is terminated after some 10 minutes by addition of an inhibitor followed by OD measurement at two wave lengths and calculation of xcex2-galactosidase activity. These tests overcome the long timespan problem of the Ames test, however, have their own disadvantages. The sensitivity is low and detection times are still a lengthy 7-8 hours. In particular the detection sensitivity of nitroarenes and polycyclic aromatic hydrocarbons is low. In addition the detection method requires a large number of actions and additions of various reagents thereby rendering the method complicated and expensive. Due to the fact that the cell has to be disrupted in order to carry out detection of any induction it is only possible to carry out one measurement on the cell.
EP-A-0.649.905 discloses that some of the disadvantages of the aforementioned tests can be overcome by placing an SOS gene upstream of a gene expressing luciferase activity such that luciferase expression occurs simultaneously with expression of the SOS gene. Subsequently a mutagenic substance can be detected or measured in a short time by measuring the luminescence. Any SOS gene is stated as being useful and the umuD,C gene (as used in the umu test) is illustrated in the examples. The sensitivity of this test is stated to be increased in comparison to that of the conventional tests i.e. the SOS chromotest and the umu test because of the smaller sample volume required for detection measurement. No relevance is attached to the promoter to be used other than the fact it must be SOS inducible. As luminescence production is immediate the measurement can occur earlier than with the lac system thereby shortening the detection time.
In WO 94/13831 DuPont also disclose the use of stress inducible promoters in combination with a luminescence gene complex to provide a genetically engineered microorganism. They state xe2x80x9calthough stress responses have been demonstrated to be useful in detecting the presence of various environmental insults it has yet to be linked to a sensitive easily detected reporterxe2x80x9d. DuPont provides an extensive list of stress inducible promoters known in the state of the art that could be useful according to them but to which the invention is not restricted. This list comprises promoters from the following regulatory circuits: heat shock, SOS, hydrogen peroxide, superoxide, fatty acid starvation, universal stress, resting state, stringent, catabolite activation, P utilisation and N utilisation. In the Examples they use the heat shock regulated protein promoters dnaK and grpE, the SOS regulated promoters recA and uvrA, the oxidative damage regulated promoters katG and micF, the universal stress promoter uspA, the stationary phase promoter xthA, the his promoter from the amino acid starvation circuit, the Lac promoter involved with the carbon starvation circuit, the phoA promoter from the phosphate limitation circuit and the glnA promoter from the nitrogen limitation circuit. This large number of examples from such a broad range of differently regulated promoters presumably serves to illustrate the broad applicability of their system. No preference is expressed or deducible for any particular group of promoters or any individual promoter. The only explicit reference to a specific SOS regulated promoter occurs in one of the examples (example 12 of the patent application) in which results with the SOS regulated promoter recA are presented. After exposing the microorganism to samples with the mutagen ethidium bromide at a concentration of 0.25 mg/ml an induction ratio of 1.9 was measured in one measurement after 180 minutes of the addition of mutagen. After addition of 0.5 xcexcg/ml mitomycin C, the induction ratio""s were measured after 100 minutes. Depending on the test strain used they varied between 4.7 to 20. This is apparent from Example 12 of the DuPont application.
Unexpectedly we have found a subgroup of stress induced promoters, in fact a subgroup of SOS regulated promoters which in combination with a luminescence reporter can be used in a microorganism system for assessing mutagenicity with improved results. This subgroup does not include the SOS regulated RecA promoter. This subgroup offers a number of advantages over the DuPont RecA-luciferase system and the other microbial toxicity testing sytems of the state of the art. In further testing and developing these new systems by mutating the promoters in a specific manner we were able to improve performance even more. In addition novel methods of testing were carried out. These novel methods of testing result in obtaining more and better data hitherto not described for any of the existing microbial toxicity tests.
The subject invention is directed at a recombinant nucleic acid sequence comprising an SOS regulated promoter with an induction ratio higher than 40, said promoter being operatively linked to a reporter encoding nucleic acid sequence encoding a reporter resulting in a signal that can be assayed as light production. Preferably a promoter with an even higher induction ratio, preferably higher than 50 is present in the recombinant nucleic acid sequence according to the invention. The induction ratio can e.g. be determined as disclosed by M. Schnurr et al. in Biochimie (1991) 73, 423-431. Alternatively the method disclosed by Peterson K. R. and Mount D. W. (1987), J. Mol. Biol. 193, 27-40 can be used. The disclosures are hereby incorporated by reference. The recA promoter disclosed in the state of the art in combination with luciferase does not fall within this category as the induction ratio of recA is appreciably lower. It is 11xc3x97 at 30xc2x0 C., which is the incubation temperature of the test. The induction ratio is an art recognised term and can be ascertained in a manner known per se for a person skilled in the art. In the literature Examples are given of how the induction ratio can be and has been ascertained together with numerical values for a number of promoters. The low induction ratio of recA is probably the reason why it is not particularly suited for a sensitive detection system based on promoter induction. The RecA promoter is the earliest promoter to be induced in the SOS regulated system. The signal(s) generated by metabolic defect(s) is (are) sensed by RecA protein. It also purportedly has a second function in mutagenesis in assisting DNA polymerase to bypass lesions. During the first twenty minutes after DNA damage the uvrA,B,C,D genes are activated to commence with excision repair. Then the recombinatorial repair pathway known as the RecF recombination pathway is very active during approximately 40 minutes. Finally, the SOS mutagenesis pathway involving umu D,C is induced.
The lowest level of mytomycin C that was used in the duPont system as illustrated in the above cited patent application was 500 ng/ml. No indication or suggestion of detection of lower levels is given. In the subject systems 7 ng/ml of mytomycin can be detected. This system is thus approximately 80 times more sensitive than the DuPont system has been illustrated to be. We found induction ratios higher than 2 from a concentration of 7,5 ng/ml in our system.
We illustrate for the first time on line measurement. On line measurement was not possible with the SOS or Ames test because of the disruption of the cell required. The other tests described have also only been used for single point determinations. We have now discovered it is possible to distinguish the presence of multiple mutagens in a sample and to determine the induction kinetics of such substances. It was totally unexpected that the presence of multiple mutagens would provide detectably different signals when following the luminescence development in time. One would to the contrary have expected a cumulative effect to arise. It is however clearly illustrated that the SOS regulated promoters form a group of promoters that exhibit such characteristics of regulation that the induction pattern differs sufficiently for various inducing chemicals to present differentiated detectable signals. The novel method is extremely simple to apply and the detection of signal can be automated. The novel method merely requires culturing a microorganism comprising an SOS regulated promoter, said promoter being operatively linked to a reporter encoding nucleic acid sequence, said reporter resulting in a signal that can be assayed as light production and contacting the microorganism with the sample to be tested followed by
measuring the luminescence of the culture, said measuring occurring at various points in time and
determining the signal to noise ratio at said points in time,
plotting the data, said data representing the kinetics of genotoxicity of said sample with multiple peaks being indicative of multiple genotoxicity compounds with different induction kinetics. Preferably the luminescence measurements are continuous. Preferably the method is carried out on line. The examples and concomitant figures illustrate the fact that various peaks are detectable with samples comprising multiple mutagens.
In a preferred embodiment the luminescence determination occurs continuously. This is particularly interesting from the point of view of automation as well as accuracy of determination of the presence of multiple genotoxic agents. The higher the signal to noise ratio the more sensitive the testing system is. Other factors that are of interest are the speed of induction, the degree of expression and the degree of fluctuation in signal to noise ratio. These factors can vary depending not only on the selected promoter but also on the host strain and the nature of the genotoxic agents. For practical uses the signal to noise ratio will be equal to or higher than 2 for 2 concentrations of genotoxic compound. A person skilled in the art will select the system best suited to their situation on the basis of one or a number of the aforementioned characteristics. The following examples illustrate the broad applicability of the novel biosensor systems. Results are shown of a number of the novel biosensor systems according to the invention consisting of various embodiments of the novel DNA sequences according to the invention in E. coli and in S. typhimurium strains. All the novel biosystems tested were better than the standard Ames and SOS chromotests which ar based on S. typhimurium and E. coli strains. The systems according to the invention were faster, more acccurate and more sensitive. We illustrate clearly for agents known to provide false negative results in the SOS chromotest and the Ames test that the novel systems according to the invention provide positive results where the known tests indeed provide false negative results. Specifically this is illustrated for novobiocine, sodiumazide, mitomycin C, naladixic acid, hydrogen peroxide, pyrene and phenantrene. The test systems according to the invention are particularly suited for detecting the presence of PAH""s (=polyaromatic hydrocrbons).
In addition the systems according to the invention do not require disruption in order to provide a measurement which is why they can subsequently be used for further testing and more importantly can provide a detectable signal over a period of time. Thus the induction kinetics of samples can be followed and determined.
An important embodiment of the invention lies in the fact that now for the first time a method for determining the presence of multiple genotoxic compounds in a sample has become available, said method comprising the steps of
culturing a host microorganism, said host microorganism comprising a nucleic acid sequence comprising an SOS regulated promoter, said promoter being operatively linked to a reporter encoding nucleic acid sequence encoding a reporter resulting in a signal that can be assayed as light production,
measuring the luminescence of the culture, said measuring occurring at various points in time, preferably continuously and determining the signal to noise ratio at said points in time, plotting the data, said data representing the kinetics of genotoxicity of said sample with multiple peaks being indicative of multiple genotoxicity compounds with different induction kinetics.
Due to selection of the novel DNA sequences and employment thereof in host cells rendering novel biosensor systems we have also created very sensitive and fast biosystems. The genoxicity of a sample can be ascertained within a time period of 5 minutes to two hours.
Another embodiment of the invention thus comprises a method for determining the presence of a genotoxic compound in a sample, said method comprising the steps of
culturing a host microorganism, said host microorganism comprising a nucleic acid sequence comprising an SOS regulated promoter with an induction ratio higher than 20, said promoter being operatively linked to a reporter encoding nucleic acid sequence encoding a reporter resulting in a signal that can be assayed as light production,
measuring the luminescence of the culture at multiple point in time and
determining whether the luminescence of the culture has changed, increased luminescence being indicative of the presence of a genotoxic compound. Preferably the induction ratio of the promoter will be 40 or more, suitably 50 or more. In the aforementioned method for determining the presence of a genotoxic compound in a sample, a preferred embodiment comprises measuring of luminescence at multiple points in time. Preferably the measurements are carried out continuously. Preferably in addition the steps of
determining the signal to noise ratio of luminescence at said points in time and
plotting the luminescence signal to noise data, said plot representing the kinetics of genotoxicity of said sample for determining the kinetics of genotoxicity of a sample. The aforementioned methods are preferably carried out with the biosensors according to the invention. Such biosensors comprise a host strain comprising the novel nucleic acid sequence according to the invention as defined elsewhere in this description. Preferably the host strain is a microorganism such as an E. coli or S. typhimurium strain. In particular the standard S. typhimurium Ames test strains have been applied as host strains and provide very suitable embodiments of a biosensor according to the invention. Strains according to the invention offer the attraction of being suited for both genotoxicity and toxicity testing. Examples of Ames test strains are TA98, TA100, TA102, TA104, TA1535 and TA1538 or the new S. typhimurium strains TA7001 to TA7006 and TA7046 (Gee et al., 1994). Also the Ames strains are standard strains that are acceptable world wide in the pharmaceutical industry. In the case of the biosensor according to the invention comprising an Ames strain as host cell the strain cannot only be used for genotoxicity testing according to the invention but also subsequently for the Ames test as such. Other suitable host strains that can thus become multifunctional will be obvious to a person skilled in the art.
Various embodiments of the nucleic acid sequence according to the invention suitable for application in a biosensor according to the invention are illustrated in the examples. A nucleic acid sequence comprising an SOS regulated promoter with an induction ratio higher than 40, said promoter being operatively linked to a reporter encoding nucleic acid sequence encoding a reporter resulting in a signal that can be assayed as light production is part of the invention. Biosensors according to the invention comprise such a nucleic acid sequence in any of the various embodiments disclosed herein. Such a DNA sequence is furthermore suitable for carrying out all the methods according to the invention as disclosed herein.
Suitable promoters from the recombinatorial repair promoters consist of the group RecF, RecJ, RecN, RecO, RecQ, ruv and uvrD promoters. The RecN promoter is illustrated in the examples. Another suitable promoter is the SfiA promoter which is also illustrated in the examples. In addition mutated promoters of the aforementioned group of SOS regulated promoters induced during recombinatorial repair having an induction ratio higher than 40 are also suitable embodiments. Such mutants may have increased promoter strength or regulation but it is vital the SOS regulation is not destroyed. An example of an extremely suitable mutation comprises a mutation in at least one LexA binding site, whereby at least one other LexA binding site remains active. A preferred mutation of this type will have a mutation in the at least one other LexA binding site that does not alter the wild type promoter sequence. An example of this type of mutant is the RecN mutant with sequence id no 7 (RecN1-3). The type of mutant with LexA mutation is particularly useful in the method of determining presence of multiple genotoxic agents and determining induction kinetics of such samples in the methods according to the invention as disclosed elsewhere in the description. Such mutants are also extremely sensitive and fast. They are particularly useful for determining the appearance and/or accumulation of genotoxic intermediate degradation or metabolic products. This can be in the field of pollutants and degradation thereof for example bioremediation of soil. This can also be useful in drug testing.
Another type of mutant that has been found to provide useful characteristics is a mutation comprising a promoter up mutation. A promoter up mutation is an art recognised term, wherein the mutation is such that the promoter sequence more closely resembles that of the consensus sequence for the RNA polymerase binding site. Such a type of mutation can comprise a mutation in the promoter xe2x88x9235 region. An example for the RecN promoter of this type of mutation is provided in the examples in the form of RecN 2-4 mutant with sequence id. no. 8. We also provide an example of a combined mutant with both LexA and promoter up mutations in the form of the RecN 3-4 mutant with sequence id no 9. In principle other improvements are possible such as mutations in the xe2x88x9210 promoter region and better spacing between the xe2x88x9235 and xe2x88x9210 region. These latter two options were not relevant for recN as this promoter already exhibits good correspondence to the "sgr"35 promoter sequence. As the xe2x88x9235 was not optimal we illustrated that a mutation rendering a closer correspondence to the "sgr"35 consensus promoter sequence resulted in ar. improvement.
In addition to the promoter sequence the nucleic acid sequence according to the invention also comprises a reporter encoding nucleic acid sequence encoding a reporter resulting in a signal that can be assayed as light production. Such sequences are known in the state of the art. A suitable embodiment is formed by a reporter encoding nucleic acid sequence comprising the luciferase A and B genes. In a preferred embodiment the sequence further comprises the luciferase C, D and E genes required for producing the limiting fatty acids substrate that is used in recycling. Details of such sequences can be found in PCT/EP patent application WO 92/15687.
A preferred method according to the invention comprises applying a microorganism suitable as Ames test strain, said microorganism further comprising a novel nucleic acid sequence according to the invention in any of the methods according to the invention described herein followed by a classical Ames test, thereby providing a method of genotoxicity mutagenicity and toxicity testing using the same strain.
Another application of the novel nucleic acid sequences according to the invention lies in use of a host microorgaanism comprising said sequence for determining toxicity. A preferred host cell is one capable of a high noise signal. For example nucleic acid sequences according to the invention with promoterup mutation as described above appear very suitable for this application. Such application can lead to IC50 calculation of toxic products. In other words a method for determining the presence of a toxic compound in a sample, said method comprising the steps of
culturing a host microorganism, said host microorganism being a host microorganism according to the invention as disclosed in any of the embodiments herein,
measuring the luminescence of the culture and
determining whether the luminescence of the culture has changed, decreased luminescence being indicative of the presence of a toxic compound is comprised within the scope of the invention.
In summary the following advantages of the invention can be given:
The possibility is created of discerning the presence of at least two genotoxic compounds presenting different induction kinetics in a sample.
The possibility is created of identifying in time the formation of intermediate genotoxic products during their metabolisation with S9 for example or degradation e.g. in bioremediation.
Kinetics of genotoxicity as well as general toxicity can be detected on line with the same cells.
A rapid test with a clear answer can be provided within a time of 5 minutes to 4 hours, preferably within 3 hours, more preferably within 2 hours.
No preliminary treatment of the cell culture prior to signal detection is required.
No disruption of the cells is required for on line detection of genotoxic compounds or general toxicity of a liquid solution.
Selected substances found to be genotoxic within 4 hours, preferably within 3 hours, more preferably within 2 hours can be further examined with the strains according to the invention for frame shift mutations or base pair substitutions with the same cells according to the Ames procedure.
There is no need for internal controls for toxicity because the decrease in basic light production is indicatoive of general toxicity.
Due to the limited test time of the experiment no strict sterile conditions are needed.
Due to the limited test time of the experiment no chemical stability tests for the test sample are needed.